Pyrimidine Nucleoside Phosphorylase Research Articles

Cloning, expression and crystallization of pyrimidine nucleoside phosphorylase fromBacillus stearothermophilus

Pyrimidine nucleoside phosphorylase (PYNP) from B. stearothermophilus has been cloned and purified for crystallization. Crystals of a potential protein-inhibitor complex have been prepared by co-crystallization techniques using the substrate analog pseudouridine. These crystals provide good-quality diffraction images to 2.7 A and belong to space group P21. The asymmetric unit contains the dimer structure of PYNP with unit-cell parameters a = 53.9, b = 71.9, c = 123.3 A and beta = 96.9 degrees.

Pyrimidine nucleoside phosphorylase and dihydropyrimidine dehydrogenase indicate chemosensitivity of human colon cancer specimens to doxifluridine and 5-fluorouracil, respectively.

We have assessed pyrimidine nucleoside phosphorylase (PyNPase) and dihydropyrimidine dehydrogenase (DPD) activity to compare the chemosensitivity of 5-fluorouracil (5-FU) and doxifluridine (5'-DFUR). Tumor samples were prepared from fresh surgical specimens of 28 patients with advanced colon carcinoma. The activity levels of the two enzymes were assessed as indicators of chemosensitivity to 5'-DFUR and 5-FU. PyNPase activity was analyzed using the HPLC method, and DPD activity was assessed according to the methods of Naguib et al. (1985). A histoculture drug response assay (HDRA) was conducted according to the methods described by Furukawa et al. (1992). The mean and standard deviation of PyNPase activity in the tumor tissue was 110 +/- 48.6 &mgr;g 5-FU/mg protein/h, which was statistically higher than the corresponding value obtained in normal tissue (60 +/- 43.1 &mgr;g 5-FU/mg protein/h) (P < 0.005). When chemosensitivity to the two drug forms was compared in 16 samples obtained from 16 cases with colon cancer, 1 specimen was sensitive to both drug forms, 3 specimens were exclusively sensitive to 5'-DFUR, 5 specimens were exclusively sensitive to 5-FU, and the other 7 specimens were insensitive to both drugs, without significance. High PyNPase activity was associated with a high chemosensitivity to 5'-DFUR, and high DPD activity correlated with a low chemosensitivity to 5-FU. However, the converse relationship was not found. We suggest that the activity of PyNPase and DPD represents a reliable indicator for the chemosensitivity of colon cancer to 5'-DFUR and 5-FU, respectively.

CHARACTERIZATION OF PYRIMIDINE NUCLEOSIDE PHOSPHORYLASE ACTIVITY IN GASTRIC CARCINOMA

Pyrimidine nucleoside phosphorylase (PyNPase) is an enzyme which catalyzes phosphorolysis of pyrimidine nucleoside, and PyNPase is mainly composed of thymidine phosphorylase (ThdPase), in human carcinoma. Recently it has been repored that ThdPase is identical to platelet-derived endothelial cell growth factor which has as angiogenic activity and ThdPase correlates with metastasis and prognosis in several tu-mors. We examined the activity of PyNPase in 85 cases of gastric carcinoma to investigate whether the activ-ity of PyNPase is related to clinicopathologic factors and prognosis. The activity of PyNPase in gastric cancer tissue is significantly higher than that is normal mucosal tissue. The activity of PyNPase is correlated with liver metastasis, ly factor and v factor. The patients with high PyNPase activity demonstrate worse prognosis than those with low PyNPase activity. Therefore careful follow-up and intensive postoperative adjuvant chemotherapy are required for patients with gastric carcinoma that has a high PyNPase activity.

The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation.

Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway. In lower organisms (e.g. Bacillus stearothermophilus) PYNP accepts both thymidine and uridine, whereas in mammalian and other higher organisms it is specific for thymidine (designated thymidine phosphorylase, TP). PYNP shares 40% sequence similarity (and presumably significant structural similarity) with human TP, which has been implicated as a growth factor in tumor angiogenesis. It is thought that TP undergoes a major conformational change upon substrate binding that consequently produces an active conformation. The crystal structure of PYNP from B. stearothermophilus with the substrate analog pseudouridine in its active site has been solved to 2.1 A resolution. This structure confirms the similarity of PYNP to TP and supports the idea of a closed active conformation, which is the result of rigid body movement of the alpha and alpha/beta domains. The active-site cleft, where the pyrimidine and phosphate substrates bind, is between the two domains. The structure reveals an asymmetric dimer in which one subunit is fully closed and the other is only partially closed. The closed conformation of PYNP serves as a good model to better understand the domain movement and overall function of TP. Active-site residues are confirmed and a possible mechanism for substrate binding and subsequent domain movement is suggested. Potent inhibitors of TP might have significant therapeutic value in various chemotherapeutic strategies, and the structure of PYNP should provide valuable insight into the rational design of such inhibitors.

Adenosine 3′:5′-Cyclic Monophosphate (cAMP)-Inducible Pyrimidine 5′-Nucleotidase and Pyrimidine Nucleotide Metabolism of Chick Embryonic Erythrocytes

Terminally differentiating erythrocytes degrade most of their RNA with subsequent release of mononucleotides. Pyrimidine mononucleotides are preferentially cleaved by an erythrocyte-specific pyrimidine 5'-nucleotidase; deficiency of this enzyme causes hemolytic anemia in humans. Details of the regulation of its activity during erythroid differentiation are unknown. The present study arose from the observation that the immature red blood cells (RBCs) of mid-term chick embryos contain high concentrations of uridine 5'-triphosphate (UTP) (5 to 6 mmol/L), which decline rapidly from days 13 to 14 onward. We analyzed two key enzymes of RBC pyrimidine nucleotide metabolism: pyrimidine nucleoside phosphorylase (PNP) and pyrimidine 5'-nucleotidase (P-5'-N), to evaluate if changes of enzyme activity during embryonic development are correlated with changes of RBC UTP. Secondly, we tested if these enzymes are under hormonal control. The results show that embryonic RBCs contain only minimal activity of PNP. In contrast, P-5'-N increases from day 13 on, suggesting that the enzyme is a limiting factor in UTP degradation. Activation of beta-adrenergic and A2A-adenosine receptors causes transcription-dependent de novo synthesis of P-5'-N. Because beta-adrenergic and adenosine receptors are also found on adult erythroid cells, P-5'-N might be an enzyme of differentiating RBCs whose expression is in part controlled by adenosine 3':5'-cyclic monophosphate (cAMP).

PyNPase expression in human colon cancer

Although colorectal cancer tissue is rich in pyrimidine nucleoside phosphorylase (PyNPase), there is no consensus as to whether cancer cells or stromal cells predominately express PyNPase. We micro-dissected OCT compound embedded frozen tissue sections into epithelial and stromal components and then analyzed the extracted samples separately. The PyNPase expression level was higher in stromal cells than in cancer cells and the difference increased with inflammation induced by the immunostimulator OK432. These results suggest that stromal cells are the major PyNPase source in colorectal cancer.

790 - Augmentation of antitumor activity of 5'-deoxy-5-f1uorourldlne by IL-12 through the up-regulation of pyrimidine nucleoside phosphorylase in murine tumor models

790 - Augmentation of antitumor activity of 5'-deoxy-5-f1uorourldlne by IL-12 through the up-regulation of pyrimidine nucleoside phosphorylase in murine tumor models

The influence of pyrimidine nucleoside phosphorylase activity on metastasis of breast cancer

The activity of pyrimidine nucleoside phosphorylase (PyNPase) was analyzed in thirty-six cases of resected breast cancer and 9 cases of resected benign breast tumor. PyNPase is an enzyme which converts 5'-DFUR to 5-FU in tumor tissue and has been reported to be identical to the platelet-derived endothelial cell growth factor (PD-ECGF) that has angiogenic activity. PyNPase activity was significantly elevated in cancer and metastatic lymph nodes compared to normal lesions of the same breast. PyNPase activity was higher in T2 and T3 breast cancer than of T1 breast cancer. Furthermore, cases of high PyNPase activity (>200 mu g FU/mg protein/h) were significantly more likely to develop distant metastasis (p<0.01) than those of low PyNPase activity. These results suggest an important role of PyNPase activity in the progression of breast cancer.

Clinical significance of pyrimidine nucleoside phosphorylase in colorectal carcinoma

The expression of pyrimidine nucleoside phosphorylase (PyNPase) mRNA in tumor (T) and normal (N) biopsy specimens obtained from 41 cases of colorectal carcinoma was examined by the reverse transcriptase/polymerase chain reaction (RT-PCR). The higher T/N ratio of the expression of PyNPase mRNA correlated significantly with the presence of lymph vessel invasion (p=0.039), the positive lymph node metastasis (p=0.014) and the advanced stage of the disease (p=0.014). There was a significant con-elation between the results determined by enzyme activity and those determined by RT-PCR (p=0.005). The findings suggested that the determination of PyNPase mRNA by RT-PCR may give useful information on tumor aggressiveness of colorectal carcinoma and this method can be used instead of enzyme activity.

Inhibition of 5'-deoxy-5-fluorouridine phosphorolysis by acyclothymidine in tumor cell homogenates.

The inhibitory effect of acyclothymidine[AcyT, 5-methyl-1-(2'-hydroxyethoxymethyl) uracil], a potent pyrimidine nucleoside phosphorylase (PyNPase) inhibitor, on 5'-deoxy-5-fluorouridine (5'-DFUR) phosphorolysis in human and mouse tumor cell homogenates was measured. Competitive inhibition was observed in MKN-74 and Lewis lung carcinoma (LLC), whereas non-competitive inhibition was observed in HeLa. The strength of the inhibitory effect by AcyT showed the following pattern: HeLa < human normal intestine < mouse normal intestine < Colon 26 < LLC < MKN-74 < DLD-1. From the kinetic parameter obtained, we simulated the inhibitory effect of AcyT on 5'-DFUR phosphorolysis in tumor cells and the intestine. These data indicated that AcyT was more sensitive in normal mouse intestine than in Colon 26 and LLC, and that orally administered AcyT can reduce the intestinal toxicity of 5'-DFUR without reducing the antitumor effect in the mouse. The present finding may have an important implication for attempts to introduce AcyT, a potent PyNPase inhibitor, into the clinic.

Uridine phosphorylase from Hymenolepis diminuta (Cestoda): kinetics and inhibition by pyrimidine nucleoside analogs.

A single pyrimidine nucleoside phosphorylase was found in the cytoplasmic extract from Hymenolepis diminuta. This enzyme preferentially cleaves uridine and, to a much lesser extent, thymidine. Its presence directly indicates the existence of pyrimidine nucleoside salvage pathway in this parasite. Detailed kinetic studies in the phosphorolytic and synthetic direction pointed to the sequential mechanism of these reactions. For phosphorolysis, Kurd = 33 microM and Kp = 806 microM. For synthesis of uridine, Kura = 204 microM and K1-P-rib. = 50 microM. Over six times higher K(m) for uracil than for uridine indicates that phosphorolysis is the favoured reaction in this tapeworm. Well known inhibitors of mammalian uridine phosphorylase: 2,2'-anhydro-5-ethyluridine and 1-(1,3-dihydroxy-2-propoxymethyl)-5-benzyluracil (DHPBU), both with Ki = 0.07 microM were potent competitive inhibitors of the enzyme from H. diminuta. The newly synthesized 2,3'-anhydro-5-ethyluridine (K. Felczak, unpublished) showed only moderate inhibitory activity (Ki = 14 microM) similarly as 1-(1,3-dihydroxy-2-propoxy-methyl)-5-benzyluracil. The same order of Ki values obtained for the investigated inhibitors vs uridine phosphorylase, irrespective whether the enzyme was isolated from rat intestinal mucosa (Drabikowska et al., 1987, Biochem. Pharmacol. 36, 4125-4128) or H. diminuta may point to a great similarity between binding sites on the parasite and the host enzyme.

Acyclic nucleosides as antiviral compounds.

Acyclovir is an effective drug for the treatment of HSV and VZV infections, which after phosphorylation to the triphosphate, inhibits viral DNA polymerase. Acyclovir has low oral bioavailability, therefore prodrugs have been developed, and the L-valyl ester, valaciclovir, recently has been licensed for the treatment of shingles. Ganciclovir is used against CMV, and famciclovir, a lipophilic prodrug of penciclovir, is marketed for shingles. The acyclic nucleoside phosphonates are active against thymidine kinase-resistant viral strains. Promising analogs are PMEA (in clinical trial for the treatment of AIDS) and (S)-HPMPC (good in vivo activity against HSV, VZV, CMV, and EBV). Oligonucleotides incorporating acyclic nucleosides at the 3'-and 5'-ends, or constituted of amino acyclic nucleosides, are resistant to cleavage by nucleases and may be useful in antisense and/or antigene therapy. HEPT is active against HIV-1: It binds in a hydrophic pocket on reverse transcriptase, rather than in the polymerase active site. Some acyclic nucleosides are potent inhibitors of purine and pyrimidine nucleoside phosphorylase. These compounds may have a therapeutic niche in combination therapy with antiviral and anticancer nucleosides, and in the treatment of diseases involving the T-cell.

Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein.

The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined. Sequence analysis showed that the genes were localized immediately downstream of the hut operon. Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp. A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure. In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine. By the use of lacZ fusions, the regulation was found to be at the level of transcription. The operon expression was subject to glucose repression. Upstream of the dra gene an open reading frame of 313 amino acids was identified. Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides. The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).

Purification and characterization of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus TH 6-2.

The purine nucleoside phosphorylase (Pu-NPase) and the pyrimidine nucleoside phosphorylase (Py-NPase) have been purified from Bacillus stearothermophilus TH 6-2. The Pu-NPase is a trimer of 30-kDa subunits and the Py-NPase is a dimer of 46-kDa subunits. The isoelectric points of Pu-NPase and Py-NPase were pH 4.3 and 4.6, respectively. The Pu-NPase could catalyze the phosphorolysis of inosine and guanosine, but not adenosine. the Py-NPase could phosphorolyze both uridine and thymidine.

Molecular cloning and expression of the pyrimidine nucleoside phosphorylase gene from Bacillus stearothermophilus TH 6-2.

The pyrimidine nucleoside phosphorylase (Py-NPase) of Bacillus stearothermophilus TH 6-2 is a dimer of 46-kDa subunits and catalyzes the reversible phosphorolysis of uridine and thymidine. The gene encoding this pyrimidine nucleoside phosphorylase (pyn gene) has been cloned and sequenced from B. stearothermophilus TH 6-2. The pyn gene corresponded to an open reading frame of 1299 nucleotides that translates into a putative 433 amino acid protein with a molecular weight of 46,271. The deduced amino terminal sequence of Py-NPase coincided with that previously found for the purified enzyme. The deduced amino acid sequence of Py-NPase shared significant similarity with those of human and Escherichia coli thymidine phosphorylases. The cloned pyn gene was overexpressed in E. coli cells to produce an active enzyme in large quantities that accounted for approximately 20% of the total protein.

Modulation of the Pharmacokinetics of 5-Deoxy-5-fluorouridine and 5-Fluorouracil in Rats by Oral Co-administration of Acyclothymidine.

The effect of an inhibitor of pyrimidine nucleoside phosphorylase (PyNPase), acyclothymidine (AcyT), on the pharmacokinetics of 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-FU) was investigated in an oral co-administration of 5'-DFUR and AcyT in rats. AcyT increased the maximal plasma concentration (Cmax) and apparent absorption rate constant (ka) of 5'-DFUR, as expected, but the increase in AUC (area under the curve) was not significant. It was expected that AcyT would only inhibit the phosphorolytic degradation of 5'-DFUR to 5-FU, but the effect was more evident on the pharmacokinetic parameters of 5-FU than on those of 5'-DFUR. AcyT also increased AUC and Cmax of 5-FU when orally co-administered with 5-FU. An inhibitory effect of AcyT on the enzymatic degradation of 5-FU in rat liver and intestinal extract was investigated. AcyT inhibited the degradation in intestinal extract but not in the liver. The result suggests that orally administered AcyT affects the pharmacokinetics of 5-FU partly by inhibiting 5-FU degradation in the process of intestinal absorption as well as by acting as an inhibitor of PyNPase.

6-Substituted and 5,6-disubstituted derivatives of uridine: stereoselective synthesis, interaction with uridine phosphorylase, and in vitro antitumor activity.

Stereoselective procedures are described for the synthesis of 6-alkyluridines by Lewis acid-catalyzed condensation of (a) trimethylsilylated 6-alkyl-4-alkylthiouracils with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (ABR) and (b) trimethylsilylated 6-alkyl-3-benzyluracils with ABR. The 4-methylthio group was subsequently removed with the use of 1 N trifluoroacetic acid and the 3-benzyl group by a new modified procedure with the use of the complex BBr3-THF. Furthermore, 6-(hydroxymethyl)uridine (39) and 5-fluoro-6-(hydroxymethyl)uridine (40) were obtained by sequential oxidation with SeO2 and reduction with tetrabutylammonium borohydride of the 6-methyl group of 6-methyluridine (5) and 5-fluoro-6-methyluridine (35), and their corresponding 6-fluoromethyl congeners 41 and 42 were obtained by DAST treatment of 39 and 40, respectively. For all the foregoing nucleosides in the fixed syn conformation about the glycosyl bond, 1H NMR spectroscopy further demonstrated that the pentose rings exist predominantly in the conformation N (3'-endo). Most of the nucleosides were weak substrates of Escherichia coli pyrimidine nucleoside phosphorylase. Enhanced susceptibility to phosphorolysis was exhibited by two of them, 39 and 41, with 6-CH2OH and 6-CH2F substituents capable of formation of an additional hydrogen bond with the enzyme. The 5-fluoro-6-substituted uridines were the poorest substrates. Cytotoxicities of the nucleosides were examined vs the human tumor cell lines MOLT-3, U-937, K-562, and IM-9, as well as PHA-stimulated human lymphocytes. Two of the analogues, 5-fluoro-6-(fluoromethyl)uridine (42) and 5-fluoro-6-(hydroxymethyl)uridine (40), exhibited cytotoxicities comparable to that of 5-fluorouracil.

Acyclothymidine alleviates intestinal toxicity of 5'-deoxy-5-fluorouridine without loss of antitumor activity in mice.

To reduce the intestinal toxicity of orally administered 5'-deoxy-5-fluorouridine (5'-DFUR) in mice, we co-administered 5'-DFUR with acyclothymidine [AcyT, 5-methyl-(2'-hydroxyethoxymethyl) uracil], a potent inhibitor of pyrimidine nucleoside phosphorylase (PyNPase). Orally administered 5'-DFUR alone caused intestinal toxicity and severe damage to the intestinal villi, while 5'-DFUR with AcyT reduced the intestinal toxicity, and prevented damage to the intestinal villi. This toxicity arising from orally administered 5'-DFUR could not be reduced by intravenous administration of AcyT, but was alleviated by oral administration. Orally co-administered AcyT showed little effect on antitumor activity of 5'-DFUR toward subcutaneously implanted Lewis lung carcinoma, though the intestinal toxicity was reduced in the tumor-bearing mice. This finding suggests that orally co-administered AcyT may prevent the undesirable conversion of 5'-DFUR to 5-FU by PyNPase during the process of absorption in the intestinal tract.

PYRIMIDINE NUCLEOSIDE PHOSPHORYLASE-ACTIVITY AND BIOLOGICAL CHARACTERISTICS OF BREAST-CANCER

We investigated whether Pyrimidine nucleoside phosphorylase (PyNPase) activity in breast cancer tissue correlated with biological characteristics of breast cancer. PyNPase activity, ER, PgR, EGFR, DNA ploidy pattern, PCNA positive cells and amplification of the c-erbB-2 gene were determined in specimens from 138 patients. PyNPase activity was significantly higher in ER negative than ER positive carcinomas (p<0.05), in PgR negative than PgR positive carcinomas (p<0.05) and significantly higher in tumors with c-erbB-2 gene amplification compared with tumors with no amplification (p<0.05). The results suggest that PyNPase activity in breast cancer tissue may be a new biological characteristic of breast cancer.

COMBINATION EFFECT OF AN ANGIOGENESIS INHIBITOR AGM-1470 WITH 5'-DEOXY-5-FLUOROURIDINE, AND WITH HORMONAL DRUGS IN DMBA-INDUCED RAT MAMMARY-TUMORS

AGM-1470, a potent angiogenesis inhibitor, is known to suppress the growth of solid tumors in vivo. In this study, we investigated the combination effect of AGM-1470 with 5'-deoxy-5-fluorouridine (5'-DFUR) which is converted to 5-FU by pyrimidine nucleoside phosphorylase, with tamoxifen (TAM), and with medroxyprogesterone acetate (MPA) in dimethylebenz(a)-anthracene (DMBA) induced rat mammary tumors. Since 5'-DFUR and MPA are noted to have antiangiogenic activity, this combination study might imply not only the addition to chemotherapeutic or hormonal drugs for the angiogenesis inhibitor but also the combination of two angiogenesis inhibitors. The combination of AGM-1470 (20 mg/kg) with 5'-DFUR (200 mg/kg) and with TAM (0.8 mg/kg) markedly suppressed the tumor growth. There was a significant difference in the suppression of tumor growth between the combination group and either drug alone group (p<0.01). No statistical combination effect between AGM-1470 and MPA was found, however MPA markedly reversed the body weight loss induced by AGM-1470. This result will be informative for clinical studies on the combination effect of AGM-1470.